FACULTY OF SCIENCE UNIVERSITY OF COPENHAGEN PhD thesis Cand.scient. Rasmus Klinck Department of Beta Cell Regeneration, Hagedorn Research Institute, and The PhD School of Science, Faculty of Science, University of Copenhagen, Denmark. The role of Hes1 in pancreas development Expression, Interdependancy and Notch signalling. Academic Advisors Dr. Olaf Nielsen, Department of Biology, Faculty of Science, University of Copenhagen – Denmark Dr. Mette C. Jørgensen, Department of Beta Cell Regeneration, Hagedorn Research Institute Denmark Submitted: 31/05/11 Cover picture: e10.5 mouse embryo expressing EGFP under the control of the Hes1 promoter manually stitched together from 15 complete image stacks. 2 Preface This Ph.D. thesis is based on experimental work performed in the Department of β Cell Regeneration at the Hagedorn Research Institute, Gentofte, Denmark from January 2008 to December 2010. The faculty supervisor on the project was Professor Olaf Nielsen, Department of Genetics, Faculty of Science, University of Copenhagen, and the project supervisor was Mette Christine Jørgensen, Ph.D., Chemist, Department of Beta Cell Regeneration at the Hagedorn Research Institute. This thesis is submitted in order to meet the requirements for obtaining a Ph.D. degree at the Faculty of Science, University of Copenhagen. The thesis is built around three scientific Manuscripts: Manuscript I: “A BAC transgenic Hes1-EGFP reporter reveals novel expression domains in mouse embryos” Submitted to Gene Expression Patterns Rasmus Klinck1, Ernst-Martin Füchtbauer2, Jonas Ahnfelt-Rønne1, Palle Serup1,Jan Nygaard Jensen1, Ole Dragsbæk Madsen1, Mette Christine Jørgensen1 1Department of Beta Cell Regeneration, Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark .2Department of Molecular Biology, Aarhus University, C. F. Møllers Alle 3 bldg.1130, DK-8000 Aarhus C, Denmark Manuscript II: “Neurog3 dependent, aberrant morphogenesis causes ectopic pancreas in Hes1 mutants” Draft manuscript that will be submitted when additional data is obtained. Rasmus Klincka, Signe Vendelbo Horn Pedersena, Heiko Lickertb, Chris V.E. Wright,c,d Jan Nygaard Jensena, Palle Serupa,d,e and Mette Christine Jørgensena* aDepartment of Beta Cell Regeneration, Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark. bHelmholtz Zentrum München, Institute of Stem Cell Research, Ingolstädter Landstraße 1 D-85764 Neuherberg Germany. cDepartment of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA dBeta Cell Biology Consortium, 2213 Garland Avenue, 9465 MRB IV, Nashville, TN 37323-0494, USA eJDRF Center for Beta Cell Therapy in Diabetes, Laarbeeklaan 103, B1090 Brussels, Belgium 3 Manuscript III: “Ptf1a is Required for Dll1 Mediated Notch Signalling in Multipotent Pancreatic Progenitors” Submitted to Genes and Development. Jonas Ahnfelt-Rønne,1,9 Mette C. Jørgensen,1,9 Rasmus Klinck,1 Jan N. Jensen,1 Ernst-Martin Füchtbauer,2 Tye Deering,3 Raymond J. MacDonald,3,7 Chris V.E. Wright,4,7 Mark A. Magnuson,5,6,7 Ole D. Madsen,1,7,8 and Palle Serup1,7,8,* 1Department of Developmental Biology, Hagedorn Research Institute, Niels Steensens Vej 6, DK-2820 Gentofte, Denmark 2Department of Molecular Biology, University of Aarhus, Aarhus, Denmark 3Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA 4Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA 5Center for Stem Cell Biology, Vanderbilt University School of Medicine, Nashville, TN, USA 6Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA 7Beta Cell Biology Consortium, 2213 Garland Avenue, 9465 MRB IV, Nashville, TN 37323-0494, USA 8JDRF Center for Beta Cell Therapy in Diabetes, Laarbeeklaan 103, B1090 Brussels, Belgium 9These authors contributed equally to this work Manuscript I and Manuscript III have recently been submitted to scientific journals. Manuscript II is based on my own data but additional data will be added from other contributors before submission to a scientific journal. However to keep the flow of this thesis I have added the section as a finished draft manuscript. In addition to the three manuscripts, this thesis contains a general introduction to the field, including manuscript specific background sections to place the thesis subject in relation to the existing knowledge within the field. A separate section with the thesis objectives is presented before the three manuscripts. Within each manuscript, the reader is presented with an overview of the results in relation to the existing knowledge and also discussions of the data. Finally I have included a general discussion of the results presented in the thesis, including conclusions and perspectives for future research within the field. Here I have tried not to overlap with the discussions in the manuscripts Rasmus Klinck, May 2011, Copenhagen, Denmark 4 Resumé (Danish summary) Diabetes er ved at blive en verdensomspændende epidemi, hvilket til dels skyldes bedre levestandard i store dele af verden hvor der i øjeblikket sker en øget industrialisering. Selvom diabetes kan behandles med et bredt udvalg af bl.a. insulin præparater, vil der med tiden altid opstå komplikationer og følgesygdomme i forbindelse med diabetes. I gennemsnit forkorter diabetes levetiden med seks år i Vesteuropa. Derfor søger forskere rundt om i verden stadig efter en kur eller en behandling der kan regulere blod-glukose niveauet endnu bedre end man kan med de nuværende behandlinger. Set fra et grundvidenskabeligt synspunkt er de mest fremadstormene strategier i øjeblikket; styret differentiering af stamceller og induceret Beta-celle regeneration. For at blive i stand til at differentiere stamceller eller opdage et medicinerbart emne, der vil føre til øget betacelle antal, er det nødvendigt at vide hvordan bugspytkirtlen udvikler sig, og hvilke netværk af faktorer der er aktive både under bugspytkirtlens udvikling og under selve sygdommen. Baseret på flere transgene muse linjer og immunohistokemiske teknikker, benytter denne afhandling sig af grundvidenskab til at afdække en lille del af det transkriptionsfaktor-netværk der er nødvendigt for korrekt udvikling af bugspytkirtlen. Arbejdet fokuserer på den transkriptionelle repressor, Hairy Enhancer of Split 1 (Hes1), i relation til formationen af bugspytkirtlen og dens celle typer. I manuskript I bruger vi en BAC transgen muse reporter linje, Tg(Hes1-EGFP)1Hri, til at analysere Hes1 udtrykket fra embryonisk dag 7.0 (E7.0) indtil postnatal dag 8 (P8). Ved E7.0 er Hes1 udtrykt eksklusivt i den definitive endoderm. Ved E8.5 i den posterior endoderm er Hes1 udtrykt med en tydelig grænse ved det 4. somit par. Anterior er Hes1 udtrykt I den anterior-intestinale portal (AIP). Efter lukningen af mavetarmkanalen er Hes1 udtrykt i den dorsale del af mavetarmkanalen, inklusiv bugspytkirtlens epitel. Senere, ved E10.5 beskriver vi Hes1-udtrykket i både det dorsale og ventrale bugspytkirtel epitel. Ved senere stadier er Hes1 udtrykt i forskellige celletyper bland andre tarm, nyre, lunge og bugspytkirtel, udtrykket af Hes1 medieret GFP matcher kendte Hes1 udtryksmønstre. Manuscript II fokuserer på Hes1-nul mutant fænotypen i relation til bugspytkirtlens organogenese. I Hes1-nul mutanten er bugspytkirtlen underudviklet og udviser et overskud af endokrin differentiation af primært glukagon celler. De endokrine celler er også ektopiske og observeres langs med den dorsale mave hvor de strækker sig i en anterior retning fra bugspytkirtlen. Den pro-endokrine faktor Neurog3 er nødvendig for at videregive den afvigende morfogenese som er styret af umådeholden Neurog3-medieret migration og ikke en patterning defekt. Manuscript III diskuterer Hes1 i relation til den velbeskrevne Notch signaleringskaskade. Det er blevet dokumenteret at Notch-signalering og nedenstrøms-genet Hes1, regulerer bugspytkirtlens endokrine 5 udvikling, muligvis gennem lateral inhibering, hvor Neurog3-udtrykkende endokrine ophavsceller via Dll1, instruerer naboceller til ikke at blive til endokrine celler. Selvom Notch er aktiv under den tidlige specificering af bugspytkirtlen, uddyber vi på forholdet mellem Hes1, Dll1 og Ptf1a. Vi viser at Dll1 udtrykket er afhængig af Ptf1a i de multipotente bugspytkirtel ophavsceller (MPC). At Hes1 er nødvendig for Dll1 og Ptf1 udtrykket i et kort tidsvindue samt at Hes1 udtrykket er uafhængigt af Dll1 medieret Notch signalering frem til E10.5. Samlet giver disse resultater ny indsigt i udtrykket og funktionen af Hes1 under den tidlige bugspytkirtels udvikling, og giver det videnskabelige felt et nyt værktøj til at arbejde videre med funktionen af Hes1. 6 Summary Diabetes is becoming a worldwide epidemic due to increased living standards throughout the world. Although the disease is treatable with a wide variety of insulins, affordable by most, complications will inevitably arise, and on average the disease takes six years off the average life expectancy in a western European country. Therefore, the medical industry is looking for a cure or a treatment that will regulate the blood glucose levels of the diabetic patient much tighter than what is currently possible. The most prevailing strategies seen from a basic research point is currently the use of; directed differentiation of stem cells, and induced β-cell regeneration. To eventually be able to either differentiate stem cells or discover a drugable target that will lead